Chemotactic cytokines (chemokines) are proteins that attract and activate leukocytes and are thought to play a fundamental role in inflammation. Chemokines are divided into four groups categorized by the appearance of N-terminal cysteine residues (“C”-; “CC”-; “CXC”- and “CX3C”-chemokines). “CXC”-chemokines preferentially act on neutrophils. In contrast, “CC”-chemokines attract preferentially monocytes to sites of inflammation. Monocyte infiltration is considered to be a key event in a number of disease conditions (Gerard, C. and Rollins, B. J. (2001) Nat. Immunol 2, 108-115; Bhatia, M., et al., (2005) Pancreatology. 5, 132-144; Kitamoto, S., Egashira, K., and Takeshita, A. (2003) J Pharmacol Sci. 91, 192-196).
The MCP family, as one family of chemokines, consists of four members (MCP-1 to 4), displaying a preference for attracting monocytes but showing differences in their potential (Luini, W., et al., (1994) Cytokine 6, 28-31; Uguccioni, M., et al., (1995) Eur J Immunol 25, 64-68).
MCP-1 is a member of the β (C—C) subfamily of chemokines. In this family, the 2 cysteins nearest to the amino terminus are adjacent to each other (thus C—C proteins). As with many other C—C chemokines, the MCP-1 gene is located on chromosome 17 in humans. The cell surface receptors that bind MCP-1 are CCR2 and CCR5.
In the following both cDNA as well as amino acid sequences of MCP-1 are indicated:
Human MCP-1 (CCL2) (GeneBank Accession: M24545)cDNA (300 bp)SEQ ID NO: 2  1 atgaaagtct ctgccgccct tctgtgcctg ctgctcatag cagccacctt cattccccaa  61 gggctcgctc agccagatgc aatcaatgcc ccagtcacct gctgttataa cttcaccaat 121 aggaagatct cagtgcagag gctcgcgagc tatagaagaa tcaccagcag caagtgtccc 181 aaagaagctg tgatcttcaa gaccattgtg gccaaggaga tctgtgctga ccccaagcag 241 aagtgggttc aggattccat ggaccacctg gacaagcaaa cccaaactcc gaagacttga Protein (Signal Sequence in bold: 23 aa; Mature MCP-1: 76 aa)SEQ ID NO: 1KVSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCP KEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT
Consistent with it being a member of the chemokine β family, MCP-1 has been shown to chemoattract and activate monocytes in vitro at subnanomolar concentrations. Elevated MCP-1 expression has been detected in a variety of pathologic conditions that involve monocyte accumulation and activation, including a number of inflammatory and non-inflammatory disease states, like rheumatoid arthritis, atherosclerosis, asthma and delayed hypersensitivity reactions.
A number of studies have underlined in particular the crucial role of MCP-1 for the development of atherosclerosis (Gu, L., et al., (1998) Mol. Cell 2, 275-281; Gosling, J., et al., (1999) J. Clin. Invest 103, 773-778); rheumatoid arthritis (Gong, J. H., et al., (1997) J. Exp. Med 186, 131-137; Ogata, H., et al., (1997) J. Pathol. 182, 106-114); pancreatitis (Bhatia, M., et al., (2005) Am. J Physiol Gastrointest. Liver Physiol 288, G1259-G1265); Alzheimer's disease (Yamamoto, M., et al., (2005) Am. J. Pathol. 166, 1475-1485); lung fibrosis (Inoshima, I., et al., (2004) Am. J Physiol Lung Cell Mol. Physiol 286, L1038-L1044); renal fibrosis (Wada, T., et al., (2004) J. Am. Soc. Nephrol. 15, 940-948), and graft rejection (Saiura, A., et al., (2004) Arterioscler. Thromb. Vasc. Biol. 24, 1886-1890). Furthermore, MCP-1 might also play a role in gestosis (Katabuchi, H., et al., (2003) Med Electron Microsc. 36, 253-262), as a paracrine factor in tumor development (Ohta, M., et al., (2003) Int. J. Oncol. 22, 773-778; Li, S., et al., (2005) J. Exp. Med 202, 617-624), neuropathic pain (White, F. A., et al., (2005) Proc. Natl. Acad. Sci. U.S.A) and AIDS (Park, I. W., Wang, J. F., and Groopman, J. E. (2001) Blood 97, 352-358; Coll, B., et al., (2006) Cytokine 34, 51-55).
The mature form of human and rodent MCP-1 is post-translationally modified by Glutaminyl Cyclase (QC) to possess an N-terminal pyroglutamyl (pGlu) residue.
Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the intramolecular cyclization of N-terminal glutaminyl residues into pyroglutamic acid (5-oxo-proline, pGlu*) under liberation of ammonia and the intramolecular cyclization of N-terminal glutamyl residues into pyroglutamic acid under liberation of water.
The N-terminal pGlu modification makes the protein resistant against N-terminal degradation by aminopeptidases, which is of importance, since chemotactic potency of MCP-1 is mediated by its N-terminus (Van Damme, J., et al., (1999) Chem Immunol 72, 42-56). Artificial elongation or degradation leads to a loss of function although MCP-1 still binds to its receptor (CCR2) (Proost, P., et al., (1998), J Immunol 160, 4034-4041; Zhang, Y. J., et al., 1994, J. Biol. Chem. 269, 15918-15924; Masure, S., et al., 1995, J Interferon Cytokine Res. 15, 955-963; Hemmerich, S., et al., (1999) Biochemistry 38, 13013-13025).
Due to the major role of MCP-1 in a number of disease conditions, a potent diagnostic tool and an anti-MCP-1 strategy is required.
As mentioned above, compelling evidence points to a role of MCP 1 in Alzheimer's disease (AD) (Xia, M. Q. and Hyman, B. T. (1999) J Neurovirol. 5, 32-41). The presence of MCP-1 in senile plaques and in reactive microglia, the residential macrophages of the CNS, has been observed in brains of patients suffering from AD (Ishizuka, K., et al., (1997) Psychiatry Clin. Neurosci. 51, 135-138. Stimulation of monocytes and microglia with Amyloid-β protein (Aβ) induces chemokine secretion in vitro (Meda, L., et al., (1996) J Immunol 157, 1213-1218; Szczepanik, A. M., et al., (2001) J. Neuroimmunol. 113, 49-62) and intracerebroventricular infusion of Aβ (1-42) into murine hippocampus significantly increases MCP-1 in vivo. Moreover, Aβ deposits attract and activate microglial cells and force them to produce inflammatory mediators such as MCP-1, which in turn leads to a feed back to induce further chemotaxis, activation and tissue damage. At the site of Aβ deposition, activated microglia also phagocytose Aβ peptides leading to an amplified activation (Rogers, J. and Lue, L. F. (2001) Neurochem. Int. 39, 333-340).
Examination of chemokine expression in the 3×Tg mouse model for AD revealed that neuronal inflammation precedes plaque formation and MCP-1 is upregulated by a factor of 11. Furthermore, the upregulation of MCP-1 seems to correlate with the occurrence of first intracellular Aβ deposits (Janelsins, M. C., et al., (2005) J. Neuroinflammation. 2, 23). Cross-breeding of the Tg2575 mouse model for AD with a MCP-1 overexpressing mouse model has shown an increased microglia accumulation around Aβ deposits and that this accumulation was accompanied by increased amount of diffuse plaques compared to single-transgenic Tg2576 littermates (Yamamoto, M., et al. (2005) Am. J. Pathol. 166, 1475-1485).
MCP-1 levels are increased in CSF of AD patients and patients showing mild cognitive impairment (MCI) (Galimberti, D., et al., (2006) Arch. Neurol. 63, 538-543). Furthermore, MCP-1 shows an increased level in serum of patients with MCI and early AD (Clerici, F., et al., (2006) Neurobiol. Aging 27, 1763-1768).
Atherosclerotic lesions, which limit or obstruct coronary blood flow, are the major cause of ischemic heart disease related mortality, resulting in 500,000-600,000 deaths annually. Percutaneous transluminal coronary angioplasty (PTCA) to open the obstructed artery was performed in over 550,000 patients in the U.S. and 945,000+ patients worldwide in 1996 (Lemaitre et al., 1996). A major limitation of this technique is the problem of post-PTCA closure of the vessel, both immediately after PTCA (acute occlusion) and in the long term (restenosis): 30% of patients with subtotal lesions and 50% of patients with chronic total lesions will go on to restenosis after angioplasty. Additionally, restenosis is a significant problem in patients undergoing saphenous vein bypass graft. The mechanism of acute occlusion appears to involve several factors and may result from vascular recoil with resultant closure of the artery and/or deposition of blood platelets along the damaged length of the newly opened blood vessel followed by formation of a fibrin/red blood cell thrombus.
Restenosis after angioplasty is a more gradual process and involves initial formation of a subcritical thrombosis with release from adherent platelets of cell derived growth factors with subsequent proliferation of intimal smooth muscle cells and local infiltration of inflammatory cells contributing to vascular hyperplasia. It is important to note that multiple processes, among those thrombosis, cell proliferation, cell migration and inflammation each seem to contribute to the restenotic process.
In the U.S., a 30-50% restenosis rate translates to 120,000-200,000 U.S. patients at risk from restenosis. If only 80% of such patients elect repeated angioplasty (with the remaining 20% electing coronary artery bypass graft) and this is added to the cost of coronary artery bypass graft for the remaining 20%, the total cost for restenosis easily reaches into billions of dollars. Thus, successful prevention of restenosis could result not only in significant therapeutic benefit but also in significant health care savings.
Although it is not clear whether elevated MCP-1 expression is the cause or consequence of the above diseases, therapeutic benefit resulted from the application of neutralizing antibodies in a number of animal models.
So far, prior art monoclonal antibodies were screened for their ability to act as receptor antagonists. None of those target the immediate pyroglutamate carrying amino terminus of MCP-1 (=MCP1 N1pE). In this context, it is important to note that deletion of amino acids 1-8 from the N-terminal region completely destroyed MCP-1 activity, suggesting that the amino-terminal region is essential for activity.
It consequently follows, that antibodies directed against the MCP-1 N1pE can play a role not only when investigating the expression and function of MCP-1 but also in therapeutic and diagnostic applications in connection with diseases or disturbances in which MCP-1 might be involved.
In view of the above, one object underlying the present invention is to provide antibodies which are selectively binding to MCP-1 N1pE.